Cryptosporidiosis is universally recognized as one of the most serious opportunistic infections that complicates AIDS. Cryptosporidium infection begins with microbial invasion of small intestinal epithelial cells (enterocytes), in which the parasite creates a peculiar niche that sequesters itself from the intestinal lumen and the host cell's cytoplasm. After amplification, endogenous parasites escape from the parasitophorous vacuole and spread throughout the epithelial surfaces of the gastrointestinal tract. No consistently reliable chemotherapy is available for the treatment of cryptosporidiosis. The need for identifying novel drug targets is imperative. Interactions of Cryptosporidium with enterocytes remain poorly characterized. Undeniably, this parasite must depend on various host cell resources since it has lost the ability to synthesize most basic metabolites. We demonstrated that Cryptosporidium developing in enterocytes contains significant amounts of cholesterol though it lacks the genes for cholesterol biosynthesis and modification. The parasite critically relies on host cholesterol for normal development since removal of cholesterol either from the medium or host cell intracellular pools, arrests its reproduction. Our overall goal is to decipher the mechanism/s for host cholesterol acquisition by Cryptosporidium. We hypothesize that the choice of enterocytes as the major sites for parasite replication may stem from the unique cholesterol transport and synthesis activities of these cells. Cryptosporidium must be equipped to intercept host cholesterol, either provided exogenously by the plasma and/or the gut, or synthesized in enterocytes. Specific Aim 1 will define the preferential source of host derived-cholesterol for Cryptosporidium and analyze the mechanisms of cholesterol delivery from the host enterocyte to the PV. We will investigate the contribution of i) the plasma low-density lipoprotein receptor-mediated endocytosis, ii) the micellar cholesterol absorption by the permease Niemann-Pick C1-like 1, and iii) the cholesterol biosynthetic pathway, to parasite growth. We documented the presence of sterols on the parasite's plasma membrane and organelles indicating that Cryptosporidium is able to internalize host cholesterol to its cell interior. Specific Aim 2 will decipher the molecular machinery developed by Cryptosporidium to transport cholesterol from the parasite surface to organelles. We will focus on the characterization of two classes of surface-exposed sterol transporters, the ATP-binding cassette G1 transporter and Niemann-Pick C1 protein present in the parasite, for cholesterol trafficking. Our long-term objective is to provide novel approaches to treat cryptosporidiosis, based on blocking the parasite's intracellular development through cholesterol starvation. Exploration of the peculiarities of cholesterol uptake by Cryptosporidium will expose new vulnerabilities. Cryptosporidium may also provide a useful model system for throwing light on cholesterol homeostatic and dysregulatory pathways in mammalian enterocytes. PUBLIC HEALTH RELEVANCE: Cryptosporidium is one of the most troublesome agents of water-borne disease that cause life-threatening disease in HIV/AIDS patients. This parasite develops inside intestinal epithelial cells where it remains protected from the hostile gut environment and is supplied with host nutrients. We will study the mechanisms by which the parasite scavenges host cholesterol, in order to specifically interfere with the pathogen's supply of this essential lipid, thereby causing its growth arrest through starvation.